Reference is made to twelve articles listed at the end of this specification, the contents of which are incorporated by reference herein.
Our studies showed that the humoral immune response (number of plaque-forming cells formed in response to sheep red blood cells) is significantly higher in mice fed a 20 g whey protein concentrate/100 g diet than in mice fed formula diets of similar nutritional efficiency containing 20 g/100 diet of any other type of commercially available semipurified food protein, such as casein, soy, wheat, corn, egg white, fish, beef protein, Spirulina maxima, Scenedesmus algae protein, or Purina mouse chow [1].
We have further shown that the immunoenhancing activity of dietary whey protein concentrate (WPC) is related to greater production of splenic glutathione (GSH) in the whey protein-fed animals during the oxygen-requiring antigen-driven clonal expansion of the lymphocyte [2]. It was then theorized that this might reflect the ability of the lymphocytes of whey protein diet-fed mice to offset potential oxidative damage, thus responding more fully to the antigenic challenge [3,4]. In fact, the capacity of a cell to recover from an oxidative insult is considered to be represented by its ability to regenerate intracellular stores of glutathione [5].
Our studies also showed that administration of S-(n-butyl) homocysteine sulfoximine, which reduces splenic glutathione in half, significantly reduced the humoral immune response of whey protein-fed mice. This was taken as further evidence for the important role of glutathione in the immunoenhancing effect of dietary whey protein [2].
Tissue glutathione concentration may be increased by administration of gamma-glutamyl-cysteine. Glutathione increased in the kidney by about 50%, 40-60 minutes after subcutaneous (s.c.) injection in mice, returning to control values 2 hours later [6]. The administered gamma-glutamylcysteine is transported intact and serves as a substrate for glutathione synthetase [7].
Advances in amino acid sequencing of food proteins allowed us to investigate the occurrence of glutamylcysteine groups in whey protein and the possible relation to glutathione promotion. Indeed, whey protein concentrate from bovine milk contains substantial amounts of glutamylcysteine groups, unlike casein, which does not increase tissue glutathione when fed to mice [1]. The glutamylcysteine groups are located primarily in the serum albumin fraction (six groups/molecule). Glutamylcysteine groups are extremely rare in animal and plant edible proteins. Extensive search of all available data on amino acid sequencing of edible proteins reveals that the Gly-Cys group with a disulfide link is indeed limited to some of the whey protein, and to the ovomucoid fraction of egg white which contains 2 of these groups in a 30,000 mol.wt.molecule [8].
Our recent [8] data further indicate that the humoral immune response is highest in mice fed a dietary whey protein concentrate exhibiting the highest solubility (unclenatured conformation) and, more importantly, a greater relative concentration of the thermolabile bovine serum albumin (.gtoreq.10%) and immunoglobulins. In addition, the mice fed this type of whey protein concentrate exhibit higher levels of tissue glutathione. The presence in the serum albumin fraction of glutamylcysteine groups (rare in food protein.) and the specific intramolecular bond as related to the undenatured conformation of the molecule are considered to be key factors in the glutathione-promoting activity of the protein mixture.
Recent experiments in Japan [9] showed that spleen cells of BALB/c male mice fed 25 g of our undenatured whey protein concentrate (WPC) (which we call "Immunocal") per 100 g diet for 4 weeks had an increased immune response to SRBC in vitro and a higher content of L3T4.sup.+ cells (12.58.times.10.sup.6 .+-.0.50) than mice fed an isocaloric diet with 25 g. pure casein/100 g. diet (3.69.times.10.sup.6 .+-.0.50). Similarly, the spleen L3T4.sup.+ /LYt-2.sup.+ ratio was 1.36.+-.0.07 in undenatured WPC fed mice and 0.55.+-.0.07 in casein-fed controls (P&lt;0,001).